阿里红三萜酸类化合物抗肿瘤作用及分子机制研究
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摘要
本论文首先对阿里红三萜酸类化合物(Triterpene acids)的超声提取工艺进行了研究。阿里红三萜酸最佳提取工艺为超声功率245.2W、提取时间34.3min、提取温度50.0℃。进一步利用硅胶柱层析分离纯化阿里红三萜酸类化合物，最终得到的样品阿里红三萜酸含量高达74.14%，是纯化前的5.13倍。采用用高效液相-质谱联用技术对阿里红三萜酸类化合物纯化后的产物进行结构分析，确定其中三萜酸类化合物包括：去氢硫色多孔菌酸，3-酮基-去氢硫色多孔菌酸，Fomitopsins C，阿里红酸F，阿里红酸G，去氢齿孔酸，去氢齿孔酮酸。
体外实验是通过MTT法，将不同浓度的药物作用于A549细胞、U937细胞、Hela细胞和HepG2细胞后，检测结果经过线性回归，确定了阿里红三萜酸对四种细胞的增殖抑制半效应量IC50为：A54929.91μg/mL，U93771.51μg/mL，Hela56.87μg/mL，HepG242.98μg/mL；选择对药物较为敏感的人肺腺癌A549细胞株，Hoechst333258染色后，置荧光显微镜下观察细胞形态的变化，发现细胞呈现凋亡的形态；不同浓度阿里红三萜酸作用于A549细胞后，采用AnnexinⅤ-FITC&PI双染并利用FCM检测细胞凋亡情况，药物可以诱导A549细胞凋亡，早期凋亡率与晚期凋亡率均高于对照组(P<0.05)。
本实验还探讨了阿里红三萜酸类化合物诱导A549细胞发生凋亡的机制。阿里红三萜酸诱导A549凋亡的机制是一方面通过降低Bcl-2蛋白的表达，增加Bax蛋白的表达，从而促进MPTP孔开放，降低△Ψm；另一方面通过升高ROS水平，ROS水平的升高可以诱导MPTP孔开放，使△Ψm降低。△Ψm的降低将导致线粒体膜内相对高渗，内部基质膨胀，外膜破裂，释放出Cyto C。Cyto C释放入胞浆后，激活Caspase级联反应，最终导致Caspase-3的活化并诱导细胞凋亡。
阿里红三萜酸类化合物抗肿瘤作用的体内实验研究是作用于S180荷瘤小鼠，结果发现，阿里红三萜酸在小鼠体内具有较好的抑制S180肿瘤细胞生长的作用，说明阿里红三萜酸体内抗肿瘤作用良好。
Fomes Officinals Ames is a kind of commonly used drugs by Xinjiang Uygurdoctors as a non-prescription for medical treatment.Uygur doctors consider thatFomes Officinals Ames is dry and hot when used as drug，which could warm thestomach，depress gas and relieve asthma， dispelwind and eliminate dampness，diminish swelling，have diuretic effect and solute snake vonem. According to somestudies， it was found that Fomes Officinals Ames could enhance their strength，extend the time of anti-fatigue and hypoxia tolerance，and could enhance the ability ofbody response to stress. Further analyzing， processing， and purifying FomesOfficinals Ames as well as the in-depth study of the pharmacological activity of activeingredients helped to expand the drug development and utilization prospects.
The main ingredients of Fomes Officinals Ames include polysaccharides，saponins and terpenoids，among which triterpenoids content accounts for as high as16%. Former studies mostly focus on the polysaccharides of Fomes Officinals Ames，finding that it can enhance imunity and eliminate oxygen free radicals. However，there are few reports about the pharmacological activity of the triterpene acids fromFomes officinalis Ames both at home and abroad.
Firstly，the extrating process of the triterpene acids from Fomes officinalis wasstudied，during which the response surface design method was adopted to optimizethe extracting conditions. The results showed that in the condition of ultrasonic power200W， ultrasonic time30min，ultrasonic temperature40℃， the total triterpeneacids from Fomes officinalis Ames got the highest yield. After that， the responsesurface design method was adopted to optimize the extracting conditions， and theoptimum processing was concluded： ultrasonic power245.2W，extraction time34.5min， extraction temperature50℃. This model predicted total flavonoids was14.89%. Verified tests showed that in the three parallel experiments using the setexperimental conditions， the average yield was14.88%， and had a good fit with thetheoretical predictions，so this model was stable and effective， which had a certainpractical significance.
Then silica-gel column chromatography was used to do gradient elution fordiferent ratio of solvent system to purify triterpene acids from Fomes officinalisAmes， and also the qulification test would be conducted to the triterpene acidscompounds with HPLC-MS. Fomes officinalis Ames crude extractings with silica gelsample were eluted with different polar solvent to get Chloroform elution content.Then silica gel column chromatography was employed to different gradient ofChloroform and methanol(V/V100:0；98:2；95:5；9:1；8:2)with TLC to monitor，and integrate the triterpene acid compounds. This method showed that the samplepurity was as high as74.14%，5.13times of that before purified， and productrecovery rate was70.26%. The purified products were analyzed with HPLC-MS，andcomparison of HPLC and MS was made according to related literature. It wascondluded that the three triterpene acids include： Dehydrosulphurenic acid，3-keto-dehydrosulfurenic acid，Fomitopsins C，Fomefficinic acid F，Fomefficinic acidG，dehydroeburicoic acid，Dehydroebu-riconic acid.
The third part is to explore the antitumor activity of Fomes Officinals Ames.Total triterpene acids from Fomes officinalis Ames acted on A549cell in nude mice，and it was found that total triterpene acids from Fomes officinalis Ames functionedwell enough to inhibit the growth of tumor. In vitro， A549cell、Hela cell、U937celland HepG2cell were selected respectively， and MTT was adopted to detect theinhibition of total triterpene acids from Fomes officinalis Ames to theabove-mentioned cells. After the result was the IC50was determined: A54929.91μg/mL，U93771.51μg/mL，Hela56.87μg/mL，HepG242.98μg/mL. A549cell，sensitive to drug， was chosen. After total triterpene acids from Fomes officinalisAmes acted on A549cells， the cells would apoptosis with the use of microscope.With FCM， it was also observed that the drug can induce the apoptosis of A549cells.
The fourth part of the study is to investigate the molecular mechanism ofanti-tumor effect of total triterpene acids from Fomes officinalis Ames. Whenstimulated by total triterpene acids from Fomes officinalis Ames， human A549cellsproduced reactive oxygen species ROS and superoxide dismutase SOD， in order to determine the role of oxidative stress in tumor cells，the researchers found that thetotal triterpene acids from Fomes officinalis Ames could increase ROS level in humanA549cells， and reduce the content of SOD by means of concentration-dependent.Then it was observed that changes of total triterpene acids from Fomes officinalisAmes in mitochondrial membrane potential and FITC-Annexin V/PI double stainingmethod. It was found that the total triterpene acids from Fomes officinalis Ames couldelevate concentration-dependent mitochondrial membrane of A549cells and induceapoptosis. Using western blot method，it was observed the effect of total triterpeneacids from Fomes officinalis Ames to mitochondrial pathway mediated apoptosis andrelated proteins (Bcl-2，Bax，Cyto C，Caspase-3and activation the expression ofCaspase-3，Caspase-9and activation of Caspase-9) expression，determine the specificmolecular mechanisms of its anti tumor effects.The results showed that totaltriterpene acids from Fomes officinalis Ames could raise the Bax/Bcl-2the proportionby concentration-dependent manner， induced mitochondrial release of cytochrome Cand activation of Caspase-3and caspase-9to induce apoptosis in A549cells. Hence，on the one hand，total triterpene acids from Fomes officinalis Ames inhibited growthof human A549cells was to increase cellular oxidative stress injury，on the other handwas to activate the mitochondrial pathway mediated apoptosis.
体外实验是通过MTT法，将不同浓度的药物作用于A549细胞、U937细胞、Hela细胞和HepG2细胞后，检测结果经过线性回归，确定了阿里红三萜酸对四种细胞的增殖抑制半效应量IC50为：A54929.91μg/mL，U93771.51μg/mL，Hela56.87μg/mL，HepG242.98μg/mL；选择对药物较为敏感的人肺腺癌A549细胞株，Hoechst333258染色后，置荧光显微镜下观察细胞形态的变化，发现细胞呈现凋亡的形态；不同浓度阿里红三萜酸作用于A549细胞后，采用AnnexinⅤ-FITC&PI双染并利用FCM检测细胞凋亡情况，药物可以诱导A549细胞凋亡，早期凋亡率与晚期凋亡率均高于对照组(P<0.05)。
本实验还探讨了阿里红三萜酸类化合物诱导A549细胞发生凋亡的机制。阿里红三萜酸诱导A549凋亡的机制是一方面通过降低Bcl-2蛋白的表达，增加Bax蛋白的表达，从而促进MPTP孔开放，降低△Ψm；另一方面通过升高ROS水平，ROS水平的升高可以诱导MPTP孔开放，使△Ψm降低。△Ψm的降低将导致线粒体膜内相对高渗，内部基质膨胀，外膜破裂，释放出Cyto C。Cyto C释放入胞浆后，激活Caspase级联反应，最终导致Caspase-3的活化并诱导细胞凋亡。
阿里红三萜酸类化合物抗肿瘤作用的体内实验研究是作用于S180荷瘤小鼠，结果发现，阿里红三萜酸在小鼠体内具有较好的抑制S180肿瘤细胞生长的作用，说明阿里红三萜酸体内抗肿瘤作用良好。
Fomes Officinals Ames is a kind of commonly used drugs by Xinjiang Uygurdoctors as a non-prescription for medical treatment.Uygur doctors consider thatFomes Officinals Ames is dry and hot when used as drug，which could warm thestomach，depress gas and relieve asthma， dispelwind and eliminate dampness，diminish swelling，have diuretic effect and solute snake vonem. According to somestudies， it was found that Fomes Officinals Ames could enhance their strength，extend the time of anti-fatigue and hypoxia tolerance，and could enhance the ability ofbody response to stress. Further analyzing， processing， and purifying FomesOfficinals Ames as well as the in-depth study of the pharmacological activity of activeingredients helped to expand the drug development and utilization prospects.
The main ingredients of Fomes Officinals Ames include polysaccharides，saponins and terpenoids，among which triterpenoids content accounts for as high as16%. Former studies mostly focus on the polysaccharides of Fomes Officinals Ames，finding that it can enhance imunity and eliminate oxygen free radicals. However，there are few reports about the pharmacological activity of the triterpene acids fromFomes officinalis Ames both at home and abroad.
Firstly，the extrating process of the triterpene acids from Fomes officinalis wasstudied，during which the response surface design method was adopted to optimizethe extracting conditions. The results showed that in the condition of ultrasonic power200W， ultrasonic time30min，ultrasonic temperature40℃， the total triterpeneacids from Fomes officinalis Ames got the highest yield. After that， the responsesurface design method was adopted to optimize the extracting conditions， and theoptimum processing was concluded： ultrasonic power245.2W，extraction time34.5min， extraction temperature50℃. This model predicted total flavonoids was14.89%. Verified tests showed that in the three parallel experiments using the setexperimental conditions， the average yield was14.88%， and had a good fit with thetheoretical predictions，so this model was stable and effective， which had a certainpractical significance.
Then silica-gel column chromatography was used to do gradient elution fordiferent ratio of solvent system to purify triterpene acids from Fomes officinalisAmes， and also the qulification test would be conducted to the triterpene acidscompounds with HPLC-MS. Fomes officinalis Ames crude extractings with silica gelsample were eluted with different polar solvent to get Chloroform elution content.Then silica gel column chromatography was employed to different gradient ofChloroform and methanol(V/V100:0；98:2；95:5；9:1；8:2)with TLC to monitor，and integrate the triterpene acid compounds. This method showed that the samplepurity was as high as74.14%，5.13times of that before purified， and productrecovery rate was70.26%. The purified products were analyzed with HPLC-MS，andcomparison of HPLC and MS was made according to related literature. It wascondluded that the three triterpene acids include： Dehydrosulphurenic acid，3-keto-dehydrosulfurenic acid，Fomitopsins C，Fomefficinic acid F，Fomefficinic acidG，dehydroeburicoic acid，Dehydroebu-riconic acid.
The third part is to explore the antitumor activity of Fomes Officinals Ames.Total triterpene acids from Fomes officinalis Ames acted on A549cell in nude mice，and it was found that total triterpene acids from Fomes officinalis Ames functionedwell enough to inhibit the growth of tumor. In vitro， A549cell、Hela cell、U937celland HepG2cell were selected respectively， and MTT was adopted to detect theinhibition of total triterpene acids from Fomes officinalis Ames to theabove-mentioned cells. After the result was the IC50was determined: A54929.91μg/mL，U93771.51μg/mL，Hela56.87μg/mL，HepG242.98μg/mL. A549cell，sensitive to drug， was chosen. After total triterpene acids from Fomes officinalisAmes acted on A549cells， the cells would apoptosis with the use of microscope.With FCM， it was also observed that the drug can induce the apoptosis of A549cells.
The fourth part of the study is to investigate the molecular mechanism ofanti-tumor effect of total triterpene acids from Fomes officinalis Ames. Whenstimulated by total triterpene acids from Fomes officinalis Ames， human A549cellsproduced reactive oxygen species ROS and superoxide dismutase SOD， in order to determine the role of oxidative stress in tumor cells，the researchers found that thetotal triterpene acids from Fomes officinalis Ames could increase ROS level in humanA549cells， and reduce the content of SOD by means of concentration-dependent.Then it was observed that changes of total triterpene acids from Fomes officinalisAmes in mitochondrial membrane potential and FITC-Annexin V/PI double stainingmethod. It was found that the total triterpene acids from Fomes officinalis Ames couldelevate concentration-dependent mitochondrial membrane of A549cells and induceapoptosis. Using western blot method，it was observed the effect of total triterpeneacids from Fomes officinalis Ames to mitochondrial pathway mediated apoptosis andrelated proteins (Bcl-2，Bax，Cyto C，Caspase-3and activation the expression ofCaspase-3，Caspase-9and activation of Caspase-9) expression，determine the specificmolecular mechanisms of its anti tumor effects.The results showed that totaltriterpene acids from Fomes officinalis Ames could raise the Bax/Bcl-2the proportionby concentration-dependent manner， induced mitochondrial release of cytochrome Cand activation of Caspase-3and caspase-9to induce apoptosis in A549cells. Hence，on the one hand，total triterpene acids from Fomes officinalis Ames inhibited growthof human A549cells was to increase cellular oxidative stress injury，on the other handwas to activate the mitochondrial pathway mediated apoptosis.
引文
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